Passage-Dependent Biological Characteristics of Acute-Phase Human Anterior Cruciate Ligament-Derived Cells

Anterior cruciate ligament (ACL)-derived cells have emerged as a promising autologous cell source for ligament tissue engineering and graft augmentation. However, although these cells are commonly expanded in vitro prior to application, their passage-dependent biological changes have not been systematically characterized, particularly in cells obtained during the acute phase after injury. This lack of knowledge may affect the reproducibility and therapeutic reliability of ACL-derived cell-based strategies. This study investigated passage-dependent changes in human ACL-derived cells obtained during the acute phase after injury, aiming to identify the passage range that retains optimal biological properties for ligament tissue engineering and graft augmentation.

ACL remnants from 7 patients were collected within 1 month after injury. Cells were isolated using type I collagenase and expanded to passage 5. Morphology was evaluated using optical microscopy and Giemsa staining. Growth kinetics were assessed by doubling time and accumulative cell counts. Extracellular matrix (ECM)- and lineage-related genes (COL1A1, COL2A1, COL3A1, cartilage oligomeric matrix protein [COMP], SOX9, tenascin C [TNC], and α-smooth muscle actin [α-SMA]) were quantified by real-time quantitative PCR.

ACL-derived cells preserved fibroblast-like morphology up to passage 3, while passages 4-5 displayed enlarged spacing and rounding. Doubling time decreased after passage 1. Collagen gene expression (COL1A1, COL2A1, and COL3A1) declined markedly from passages 3 to 4 (p < 0.001), while COMP, SOX9, and TNC showed no significant changes. α-SMA also decreased from passage 2. Collagen gene ratios were unchanged despite reduced absolute expression.

ACL-derived cells retain favorable morphology, proliferation, and ECM-producing capacity within passages 1-3, whereas passages ≥4 exhibit observable morphological changes and a possible decline in ligamentogenic capacity. These findings indicate that maintaining expansion within early passages may be preferable for translational applications such as scaffold reseeding and biologically enhanced graft ligamentization.